Diseases caused by Salmonella bacteria range from a mild, self-limiting diarrhea to serious gastrointestinal and septicemic disease in humans and animals. Salmonella is a gram-negative, rod-shaped, motile bacterium (nonmotile exceptions include S. gallinarum and S. pullorum) that is non-spore forming. Environmental sources of the organism include water, soil, insects, factory surfaces, kitchen surfaces, animal feces, raw meats, raw poultry, and raw seafoods.
Salmonella infection is a widespread occurrence in animals, especially in poultry and swine, and is one of the most economically damaging of the enteric and septicemic diseases that affect food producing animals. Although many serotypes of Salmonella have been isolated from animals, S. chloeraesuis and S. typhimurium are the two most frequently isolated serotypes associated with clinical salmonellosis in pigs. In swine, S. typhimurium typically causes an enteric disease, while S. choleraesuis (which is host-adapted to swine) is often the etiologic agent of a fatal septicemic disease with little involvement of the intestinal tract. S. dublin and S. typhimurium are common causes of infection in cattle; of these, S. dublin is host adapted to cattle and is often the etiologic agent of a fatal septicemia disease. Other serotypes such as S. gallinarum and S. pullorum are important etiologic agents of salmonellosis in avian and other species. Although these serotypes primarily infect animals, S. dublin and S. chloeraesuis also often cause human disease.
Various Salmonella species have been isolated from the outside of egg shells, including S. enteritidis which may even be present inside the egg yolk. It has been suggested that the presence of the organism in the yolk is due to transmission from the infected layer hen prior to shell deposition. Foods other than eggs have also caused outbreaks of S. enteritidis disease in humans.
S. typhi and S. paratyphi A, B, and C produce typhoid and typhoid-like fever in humans. Although the initial infection with salmonella typically occurs through the gastrointestinal tract, typhoid fever is a systemic disease that spreads throughout the host and can infect multiple organ sites. The fatality rate of typhoid fever can be as high as 10% (compared to less than 1% for most forms of salmonellosis). S. dublin has a 15% mortality rate when the organism causes septicemia in the elderly, and S. enteritidis has an approximately 3.6% mortality rate in hospital/nursing home outbreaks, with the elderly being particularly affected.
Numerous attempts have been made to protect humans and animals by immunization with a variety of vaccines. Many of the vaccines provide only poor to moderate protection and require large doses to be completely efficacious. Previously used vaccines against salmonellae and other infectious agents have generally fallen into four categories: (i) specific components from the etiologic agent, including cell fractions or lysates, intact antigens, fragments thereof, or synthetic analogs of naturally occurring antigens or epitopes (often referred to as subunit vaccines); (ii) antiidiotypic antibodies; (iii) the whole killed etiologic agent (often referred to as killed vaccines); or (iv) an avirulent (attenuated) derivative of the etiologic agent used as a live vaccine.
Reports in the literature have shown that attenuated live vaccines are more efficacious than killed vaccines or subunit vaccines for inducing protective immunity. Despite this, high doses of live vaccines are often required for efficacy and few live-attenuated Salmonella vaccines are commercially available. Ideally, an effective attenuated live vaccine retains the ability to infect the host without causing serious disease and is also capable of stimulating humoral (antibody-based) immunity and cell-mediated immunity sufficient to provide resistance to any future infection by virulent bacteria.
Several attenuation strategies have been utilized to render Salmonella avirulent [Cardenas et al., Clin Microbial Rev. 5:328-342 (1992); Chatfield et al., Vaccine 7:495-498 (1989); Curtiss, in Woodrow et al., eds., New Generation Vaccines, Marcel Dekker, Inc., New York, p. 161 (1990); Curtiss et al., in Kohler et al., eds., Vaccines: new concepts and developments. Proceedings of the 10th Int'l Convocation of Immunology, Longman Scientific and Technical, Harlow, Essex, UK, pp. 261-271 (1987); Curtiss et al., in Blankenship et al., eds., Colonization control of human bacterial enteropathogens in poultry, Academic Press, New York, pp. 169-198 (1991)]. These strategies include the use of temperature sensitive mutants [e.g., Germanier et al., Infect Immun. 4:663-673 (1971)], aromatic and auxotrophic mutants (e.g., -aroA, -asd, -cys, or -thy [Galan et al., Gene 94:29-35 (1990); Hoiseth et al., Nature 291:238-239 (1981); Robertsson et al., Infect Immun. 41:742-750 (1983); Smith et al., Am J Vet Res. 45:59-66 (1984); Smith et al., Am J Vet Res. 45:2231-2235 (1984)]), mutants defective in purine or diaminopimelic acid biosynthesis (e.g., Δpur and Δdap [Clarke et al., Can J Vet Res. 51:32-38 (1987); McFarland et al., Microb Pathog. 3:129-141 (1987); O'Callaghan et al., Infect Immun. 56:419-423 (1988)]), strains altered in the utilization or synthesis of carbohydrates (e.g., ΔgalE [Germanier et al., Infect Immun. 4:663-673 (1971); Hone et al., J Infect Dis. 156:167-174 (1987)]), strains altered in the ability to synthesize lipopolysaccharide (e.g., galE, pmi, rfa) or cured of the virulence plasmid, strains with mutations in one or more virulence genes (e.g., invA) and mutants altered in global gene expression (e.g., -cya -crp, ompR or -phoP [Curtiss (1990), supra; Curtiss et al. (1987), supra; Curtiss et al. (1991)], supra).
In addition, random mutagenesis techniques have been used to identify virulence genes expressed during infection in an animal model. For example, using a variety of approaches, random mutagenesis is carried out on bacteria followed by evaluation of the mutants in animal models or tissue culture systems, such as Signature-Tagged Mutagenesis (STM) [see U.S. Pat. No. 5,876,931].
However, published reports have shown that attempts to attenuate Salmonella by these and other methods have led to varying degrees of success and demonstrated differences in both virulence and immunogenicity [Chatfield et al., Vaccine 7:495-498 (1989); Clarke et al., Can J Vet Res. 51:32-38 (1987); Curtiss (1990), supra; Curtiss et al. (1987), supra; Curtiss et al. (1991), supra]. Prior attempts to use attenuation methodologies to provide safe and efficacious live vaccines have encountered a number of problems.
First, an attenuated strain of Salmonella that exhibits partial or complete reduction in virulence may not retain the ability to induce a protective immune response when given as a vaccine. For instance, ΔaroA mutants and galE mutants of S. typhimurium lacking UDP-galactose epimerase activity were found to be immunogenic in mice [Germanier et al., Infect Immun. 4:663-673 (1971), Hohmann et al., Infect Immun. 25:27-33 (1979); Hoiseth et al., Nature, 291:238-239 (1981); Hone et al., J. Infect Dis. 156:167-174 (1987)] whereas Δasd, Δthy, and Δpur mutants of S. typhimurium were not [Curtiss et al. (1987), supra, Nnalue et al., Infect Immun. 55:955-962 (1987)]. All of these strains, nonetheless, were attenuated for mice when given orally or parenterally in doses sufficient to kill mice with the wild-type parent strain. Similarly, ΔaroA, Δasd, Δthy, and Δpur mutants of S. chloeraesuis were avirulent in mice, but only ΔaroA mutants were sufficiently avirulent and none were effective as live vaccines [Nnalue et al., Infect Immun. 54:635-640 (1986); Nnalue et al., Infect Immun. 55:955-962 (1987)].
Second, attenuated strains of S. dublin carrying mutations in phoP, phoP crp, [crp-cdt] cya, crp cya were found to be immunogenic in mice but not cattle [Kennedy et al., Abstracts of the 97th General Meeting of the American Society for Microbiology. B-287:78 (1997)]. Likewise, another strain of S. dublin, SL5631, with a deletion affecting gene aroA was highly protective against lethal challenge to a heterologous challenge strain in mice [Lindberg et al., Infect Immun. 61:1211-1221 (1993)] but not cattle [Smith et al., Am J Vet Res. 54:1249-1255 (1993)].
Third, genetically engineered Salmonella strains that contain a mutation in only a single gene may spontaneously mutate and “revert” to the virulent state. The introduction of mutations in two or more genes tends to provide a high level of safety against restoration of pathogenicity by recombination [Tacket et al., Infect Immun. 60:536-541 (1992)]. However, the use of double or multiple gene disruptions is unpredictable in its effect on virulence and immunogenicity; the introduction of multiple mutations may overattenuate a bacteria for a particular host [Linde et al., Vaccine 8:278-282 (1990); Zhang et al., Microb. Pathog., 26(3):121-130 (1999)].
Of interest to the present invention is the identification of pathogenicity islands (PAIs) in Salmonella and other bacteria, which are large, sometimes unstable, chromosomal regions harboring clusters of genes that often define virulence characteristics in enteric bacteria. The DNA base composition of PAIs often differs from those of the bacterial chromosomes in which they are located, indicating that they have probably been acquired by horizontal gene transfer. One Salmonella pathogenicity island containing genes required for epithelial cell invasion has been identified at around 63 centisomes (cs) on the S. typhimurium chromosome, and has been shown to contain genes encoding components of a type III (contact-dependent) secretion system, secreted effector proteins, and associated regulatory proteins [Millis et al., Mol Microbiol 15(4):749-59 (1995)] A second Salmonella PAI of 40 kb is located at 30.7 and has been designated Salmonella pathogenicity island 2 (SP12) [Shea et al., Proc. Nat'l Acad. Sci. USA, 19;93(6):2593-2597 (1996)]. Nucleotide sequence analysis of regions of SPI2 revealed genes encoding a second type III secretion apparatus that has been suggested to be involved at a stage of pathogenesis subsequent to epithelial cell penetration. Mutations in some genes within SPI2 have been shown to result in attenuation of bacterial virulence in mice. See U.S. Pat. No. 5,876,931; Shea et al., Proc. Natl. Acad. Sci. USA, 93:2593-2597 (1996); Ochman et al., Proc Natl Acad Sci USA, 93(15):7800-7804 (1996); Hensel et al., J. Bacteriol., 179(4):1105-1111 (1997); Hensel et al., Molec. Microbiol., 24(1):155-167 (1997); Dunyak et al., poster presented at 97th General Meeting of the American Society for Microbiology (1997), p. 76.
A need continues to exist for more safe and efficacious live attenuated Salmonella vaccines that ideally do not need to be administered at very large doses.